We are focusing our studies 1. on proving or disproving the hypothesis that TRPC type cation channels are, together with Orai molecules, components of the channels that mediate of store depletion activated calcium entry;and 2. on determining their roles in health and disease. In earlier studies we had discovered six of the seven TRPC channels, cloned full length cDNAs of four (TRPC1, TRPC2, TRPC3 and TRPC6) and shown them to be activated by maneuvers that stimulate the Gq-PLCb-IP3 mediated depletion of calcium stores, and showed that peptides of the IP3 receptor that interact in vitro with TRPC3 segments (GST pull-down) affect store depletion activated calcium entry, also store operated calcium entry or SOCE. But direct activation of TRPC3 upon thapsigargin stimulated store depletion independent of G protein-PLC-beta activation failed to show the classical store operated calcium entry response. Moreover, electrophysiological measurements only revealed the appearance of non-selective cation channels when TRPCs were expressed in model cells, which, while permeant to Ca, lacked the required Ca selectivity exhibited if they were the sole responsible molecules forming the Icrac channels. During the last three years, we focused on the role of the newly discovered Orai molecules in SOCE. Others showed that co-expression of Orai plus an also newly discovered membrane protein, STIM, which is the calcium sensor and responsible for activation of plasma membrane SOCE channel complex, results in giantSOCE (also monsterSOCE). These other studies also showed that mutations in Orai changed the permeation properties of SOCE and Icrac leading to the new, 2006-2007 proposal that Orai is the SOCE channel proper without involvement of TRPC channels. However, previous data connecting TRPCs to SOCE, while not conclusive, had clearly indicated that TRPCs are at least in part involved in SOCE. During the previous two years we discovered that Orai is able to confer the so far missing store depletion responsiveness to store-insensitive TRPCs stably expressed in HEK cells and, in collaboration with Drs. David Armstrong and Christian Erxleben from the Laboratory of Neurobiology, that co-expression of Orai and TRPC leads to reconstitution of TRPC-dependent Icrac. We are also performing a general screening to determine which TRPCs can be shown to interact functionally with Orai. So far we have found that TRPC1, C3, C6, and C7 become sensitive to store depletion when provided with Orai. New stable cell lines have been generated that express TRPC3 and Orai. With these cells we hope to be able, by crosslinking, to characterize the interaction of TRPCs with Orai in intact cells. If this were successful, we would go on to map the interaction surface between Orai and TRPC. ,Finally, and importantly, we found that an Oria mutant (Orai-R91W) has dominant negative properties and interferes not only with store operated Ca entry and receptor operated Ca entry, but also with diacylglycerol-activated Ca entry which is mediated by TRPCs 3 and 6, and does not involve either store depletion or PLC activation. This last finding strengthened our belief that TRPCs and Orai form functional units. As a second approach to answer the question whether TRPC channels are essential in SOCE we have begun a program in which by breeding generate compound knockout mice, i.e. mice deficient in ever more TRPC channels. In previous years we had generated mice deficient in TRPC1, TRPC3 and TRPC6 which have been used to study physiological roles of TRPC channels. During this last year we added TRPC5 and TRPC7 to this list. We are collaborating with Prof. Veit Flockerzi (Homburg (Saarland), Germany) to create compound KO mice. Between the two laboratories we have generated C1-C3-C6 and C1-C4-C6 triple KO mice. These mice breed well. Importantly for our project to determine the composition of the SOCE channel, embryonic fibroblasts from the either of these two triple KO mouse strains express only one TRPC: TRPC2. We are currently engaged in down-regulating TRPC2 to establish whether a TRPC is essential for SOCE. Other collaborations, based on the availability of TRPC deficient mice, are exploring roles of TRPCs in hearing, in circadian rhythms, in slow postsynaptic currents of glutamate synapses involved in learning and memory, and in hypertension. Thus, our KO mice will serve to further investigate the physiological roles of the TRPC family of TRP channels. One focus of interest is the role of TRPCs in excitotoxicity. Preliminary data from others have shown that TRPC KO mice are more resistant to the deleterious effects of electroshock, or massive activation of excitatory receptors with glutamic acid.